One of the most striking and ancient examples of small RNA regulation of gene expression is the process of RNA editing in the mitochondria of trypanosomes. In these parasites, RNA editing involves extensive uridylate insertions and deletions within most of the mitochondrial mRNAs. Over 1200 small guide RNAs (gRNAs) are predicted to be responsible for directing the sequence changes that create start and stop codons, correct frameshifts and for many of the mRNAs, generate most of the open reading frame of the mRNA. In addition, alternative editing appears to be extensive and creates the opportunity for unprecedented protein diversity. Guide RNAs are key components of the RNA editing process; information on the total number and transcript levels of the gRNAs involved in this complex process would provide key information necessary for our understanding of its regulation. Despite many conventional sequencing attempts, large numbers of the gRNAs needed for the extensive editing of the protein genes are still unidentified. In addition, no information on gRNA expression levels exists. The recent development of second generation, ultra-high-throughput sequencing platforms allow both the identification of rare transcripts and the quantification of specific RNA levels based on the density of corresponding reads. The objective of this proposal is to utilize deep sequencing technology to characterize the full gRNA transcriptomes in the two main life cycle stages of Trypanosoma brucei. High-throughput sequencing will allow the identification of all gRNAs within a population, provide quantitative information on their expression levels and determine the transcriptional structure of the gRNA genes. Characterization of the full complement of gRNAs needed for both conventional and alternative editing, will allow the identification of the multiple target sites within each mRNA. With this information we can begin to predict the free energy requirements for the multiple gRNA/mRNA interactions necessary for the editing of a single transcript. These data will provide insight into both the developmental regulation of editing and the selective use of alternative guide RNAs. In addition, knowledge of the transcriptional structure of gRNAs may allow the identification of specific minicircle sequence elements important for transcription and processing. Small RNAs are now known to be key regulators of gene expression. The ability of small RNAs to accurately and efficiently basepair with their specific targets is crucial to their function. Dissection of the targeting strategies used in this ancient RNA editing system, may provide insight into the evolution of small RNA targeting strategies. PUBLIC HEALTH RELEVANCE: The kinetoplastid protozoa include the causative agents for three of the most severe parasitic diseases targeted for attention by the World Health Organization (WHO); Leishmaniasis, sleeping sickness and Chagas disease. Together these parasites affect approximately 550 million people in the developing world. RNA editing is essential for mitochondrial function and is unique to these parasites making this process a good target for drug intervention.